We have established and provided preliminary characterization of 16 stable murine monoclones that produce antibodies specific for human DNA polymerase-alpha, three of which possess potent neutralizing activity. Several of the antibodies have been used to develop rapid, reproducible immunopurification protocols that yield DNA polymerase-alpha fractions of extraordinarily high specific activity and purity from feasible quantities of cultured cells in only 3 days. These fractions contain an interesting and novel set of polypeptides, including a family of large polypeptides that range between 20 and 130 kilodaltons. The epitopes recognized by all of our monoclonal antibodies map within this family of large polypeptides, which have almost identical tryptic maps; and we have thus tentatively concluded that within this family of proteins resides the polymerase-alpha catalytic activity. We have documented the presence of a DNA primase activity in the immunoaffinity-purified polymerase-alpha fraction. We have characterized that primase activity in detail and have published an entirely novel model of primase activity that posits the tightly synchronized coupling of that activity with polymerase-alpha and proposes a solution to the important problem of the switching event by which primer synthesis is coupled to DNA replication. Because we cannot dissociate this purified DNA polymerase-alpha/DNA primase holoenzyme complex, we are initiating a new series of monoclonal antibody approaches using the purified holoenzyme fraction as antigen and rats as recipients. This approach will develop a second generation of monoclones with specificities for the individual peptide components of the holoenzyme complex and, particularly, with specificity for the DNA primase activity. By this approach, we plan to dissect these two principal catalytic activities and obtain further insight into their protein structure, mechanisms of catalysis, and modes of interaction. We are also well along in a major study based on our monoclonal antibody specificities and cross-reactivities that will establish the chromosomal localization of human DNA polymerase-alpha. Data in hand not only identify the chromosome but focus on a small chromosomal segment that contains known protein markers that should permit establishment of polymerase-alpha linkage relationships and, more important, the isolation of a chromosomal subsegment containing polymerase-alpha (and perhaps primase) in an enriched form that will provide an invaluable resource for molecular genetic identification, amplification, and manipulation of polymerase-alpha sequences. (I)